RU2199317C2 - Method for decreasing sickness rate of necrotizing enterocolitis in children (versions) - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: child predisposed to necrotizing enterocolitis is introduced with, at least, one n-6 polyunsaturated fatty acid (PUFA), for example, arachidonic acid, in combination with, at least, one n-3 PUFA, for example, docosahexaenic acid. The mentioned PUFA are usually used in the form of either phospholipid or phosphatidylcholine. The mentioned injection is carried out either enterally or parenterally. Combination of mentioned PUFA provides synergistic effect in the treatment of necrotizing enterocolitis. According to other variants of the present innovation, a child is introduced with efficient amount of phospholipids, predominantly obtained out of egg lecithin, or efficient quantity of choline is introduced, predominantly, phosphatidylcholine. Application of mentioned preparations decreases sickness rate of necrotizing enterocolitis which is considered to be very dangerous for juvenile life. EFFECT: higher efficiency. 28 cl, 7 ex, 7 tbl

Description

FIELD OF THE INVENTION
The invention relates mainly to enteral formulations that contain long chain polyunsaturated fatty acids (PUFAs), and a method for producing these enteric compositions, as well as methods for reducing the incidence of necrotic enterocolitis. In particular, this invention relates to enteral compositions containing long-chain PUFAs — arachidonic acid (AA) and docosahexaenoic acid (DHA), which are substantially free of cholesterol and can be obtained from egg yolk lipids. Long-chain PUFAs derived from egg yolk are primarily in the form of phospholipids. The method of obtaining this composition provides improved organoleptic properties and stability. Enteral administration of these compositions may reduce the incidence of necrotic enterocolitis.

State of the art
Long-chain PUFAs in formulations or compositions for enteral use are the subject of widespread discussion in the literature. For example, US Pat. No. 4,670,285 (Clandinin) discloses a specific fat mixture suitable for use in infant formula. In particular, the fat mixture in Clandinin contains at least one C ₂₀ or C ₂₂ n-6 fatty acid and a C ₂₀ or C ₂₂ n-3 fatty acid. It is described that these fatty acids in certain certain amounts avoid the harmful effects of a mixture of fats in baby food. C ₂₀ or C ₂₂ n-6 fatty acids are present in the product in a total amount of about 0.13 to 5.6 wt.% Of all fatty acids in the product. With ₂₀ or C ₂₂ n-3 fatty acids (if any) comprise from about 0.013 to 3.33 wt.% Of the total amount of all fatty acids in the product. Clandinin opens up the use of egg lipids as a source of n-6 and n-3 fatty acids, but the egg lipid used in Clandinin also contains high cholesterol. Further, this document recommends the use of 75 to 95 parts (by weight) of egg yolk lipid, while the rest of the oils are coconut oil or soybean oil. The nomenclature used in clandinin for fatty acids will be used in this context.

WO 93/20717 discloses a baby food composition that contains no more than sub-irritant amounts of long chain free fatty acids (C ₁₆ -C ₂₂ ) and triglycerides. This application also describes that the preparation of lower alkyl esters, such as ethyl esters of these fatty acids, in baby food formulations virtually eliminates the ability of fatty acids to destroy the intestinal epithelium of a child, but provides absorption and processing of fatty acid structures.

 US patent 4918063, issued by Lichtenberger, discloses compositions containing unique mixtures of phospholipids and neutral lipids for the prevention and treatment of ulcers and inflammatory bowel diseases. This patent describes mixtures of saturated or unsaturated phospholipids with saturated or unsaturated triglycerides and / or sterols, which provide effective protection against ulcers in experimental animal models. This patent also describes the inclusion of multivalent cations or antioxidants in the lipid mixture to increase activity.

 In International Publication WO 96/10922 Kohn et al. describe a mixture of fats of an infant formula, characterized in that arachidonic acid and docosahexaenoic acid are present in the mixture of fats in the form of phospholipids.

 In European Patent Application 0376628 B1, Tomarelli describes a vegetable oil-only fat composition that uses non-standard palm oil or non-standard palm oleic oil as the sole source of palmitic acid. It is also described that all fatty compositions based on vegetable oils are particularly suitable for infant formula for premature (or low birth weight) babies. Fat compositions for premature babies from Tomarelli's application include medium chain triglycerides (TSC) with non-standardized oil containing palmitic acid, oil containing lauric acid, oil containing oleic acid, and oil containing linolenic acid.

 Despite the important contribution of the materials discussed above, there remains a need for baby food formulations that contain egg phospholipids as a source of long-chain PUFAs in concentrations suitable for nutrition. In addition, there is also a need to develop methods for preparing enteric formulations containing egg phospholipids so that the composition has acceptable organoleptic properties. These compositions, in particular, are used in baby food formulations for term-born and premature babies, for which the need for long-chain PUFAs for the proper development of the nervous system and the development of visual activity has been established. In addition, they may have a protective effect on the intestines.

 Necrotic enterocolitis (NEC) is a serious problem for children with a birth weight of less than approximately 1500 g. Despite studies for almost three decades, the exact etiology and pathophysiology of NEC remains unclear. NEC is a life-threatening disease characterized by ischemic necrosis of the structures of the digestive tract involved and intestinal pneumatosis, which often leads to intestinal perforation. Premature infants with NEC have a clinical picture characterized by temperature instability, drowsiness, stagnation in the stomach, vomiting, bloating, large amounts of blood or the presence of occult blood in the stool and radiographic confirmation of intestinal pneumatosis, air in the portal veins or pneumoperitoneum. Apnea appears, shock and subcutaneous adiponecrosis quickly occur, and death usually occurs.

 Many authors have made various observations and presented the factors that influence this disease. (Nue, Pediatr. Clin. North. Am., April 1996, issue 43 (2), pp. 409-32). Examples are the following observations and factors.

 Plageole et al. Necrotic enterocolitis in newborns, a review for clinicians. Union-Med-Can., 1991, Sept. - Oct., vol. 120 (5), p. 334-8. The authors suggest that the pathogenesis of NEC includes mesenteric ischemia, gastrointestinal maldevelopment, enteral feeding, and possibly even infection.

Caplan et al. The role of platelet activation factor and tumor necrosis factor-α in neonatal necrotic enterocolitis. Journal of Pediatrics, June 1990, p. 960-964. The authors show that platelet activation factor and tumor necrosis factor-α levels are elevated in patients with NEC.
Kliegman et al. Clostridia as pathogens in neonatal necrotic enterocolitis, The Journal of Pediatrics, August, 1979, p. 287-289. The authors describe the isolation of Clostridia perfringens in children with neonatal NEC.

 Ostertag et al. Early enteral feeding does not affect the incidence of necrotic enterocolitis. Pediatrics, vol. 77, No. 3, March 1986, p. 275-280. The authors show that diluted early enteral administration of high-calorie foods does not adversely affect the incidence of NEC.

 Rell et al. Neonatal necrotic enterocolitis. Annals of Surgery, vol. 187, January 1978, No. 1, p. 1-7. The authors suggest the use of combination antimicrobial therapy to treat children with NEC.

 Eyal et al. Necrotic enterocolitis in babies with very low birth weight: a comparison of breastfeeding with parenteral feeding. Archives of Disease in Childhood, 1982, no. 57, p. 274-276. The authors show that the incidence of NEC in children with low body weight at birth decreased with a delay in the start of enteral feeding.

 Finer et al. Vitamin E and necrotic enterocolitis. Pediatrics, vol. 73, No. 3, March, 1984. The authors suggest that the administration of vitamin E to reduce the incidence of acute complications after retrolental fibroplasia may be associated with an increase in the incidence of NEC.

 Brown et al. Prevention of necrotic enterocolitis in newborns. JAMA, November 24, 1978, v. 240, No. 22, p. 2452-2454. The authors show that NEC can be avoided by using a slow-increase feeding regimen.

 Kosloske. Pathogenesis and prevention of necrotic enterocolitis: a hypothesis based on personal observations and a review of the literature. Pediatrics, vol. 74, No. 5, December, 1984, p. 1086-1092. The author hypothesizes that NEC occurs when two or three pathological events coincide: (1) intestinal ischemia, (2) colonization by pathogenic bacteria, and (3) excess protein substrate in the intestinal cavity.

 Kosloske, supra shows that NEC rarely occurs in children fed only breast milk. In humans, breast milk plays a role in passive immunization of the intestines of newborns and contains factors that contribute to the growth of Bifidobacterium in the intestinal flora. It has also been shown that freezing, pasteurization or storage have an adverse effect on the beneficial content of human milk.

 Thus, there are many different opinions regarding the etiology and treatment of NEC and there remains a need to create compositions and methods that can better treat and / or reduce the frequency of this dangerous and often fatal condition.

SUMMARY OF THE INVENTION
The present invention has many aspects. In a first aspect, the invention provides a method for reducing the incidence of necrotic enterocolitis in children susceptible to necrotic enterocolitis. The method comprises administering an effective amount of at least one long-chain PUFA selected from the group C ₂₀ n-6 fatty acids, C ₂₂ n-6 fatty acids, C ₂₀ n-3 fatty acids and C ₂₂ ω-3 fatty acids acids. For example, the effectiveness of the administration of arachidonic acid, an n-6 fatty acid, has been shown. Administration may be enteral or parenteral. A combination of n-6 and n-3 fatty acids is preferred, for example, the combined use of arachidonic acid and docosahexaenoic acid. The level of enteral administration is at least 1.0 mg of n-6 fatty acids per kilogram of body weight per day. In a more preferred embodiment, a combination of n-6 and n-3 fatty acids is used in a weight ratio of from about 2: 1 to about 4: 1 with the introduction of at least 5.0 mg of long chain n-6 fatty acids in a day.

 The method can be carried out by administering with food a sufficient amount of an enteric composition containing arachidonic acid and docosahexaenoic acid, with the aim of delivering from about 1.0 to about 60 mg per kg per day of arachidonic acid to the child and from about 0.25 to about 35 mg per kg per day of docosahexaenoic acid. More typical amounts are from about 5.0 to about 40 mg of arachidonic acid and from about 1.5 to about 20 mg of docosahexaenoic acid per kg per day. Preferably, the weight ratio of arachidonic acid and docosahexaenoic acid is in the range of from about 2 to about 4. It is preferred that the long chain polyunsaturated fatty acids are in the form of phospholipids, especially phosphatidylcholine. These phospholipids are present in high concentrations in egg lecithin and egg phosphatides.

 Thus, in a further aspect, the invention provides a method for reducing the incidence of necrotic enterocolitis in children. This method involves administering to this child a sufficient amount of an enteral nutritional composition containing protein, carbohydrate and phospholipids to provide at least 1.0 mg of long chain n-6 polyunsaturated fatty acids per day. Preferably, the feeding further provides at least 0.5 mg of long chain n-3 polyunsaturated fatty acids per day as arachidonic acid and docosahexaenoic acid, respectively.

 In another aspect, the invention provides a method of reducing the incidence of necrotic enterocolitis in children. This method involves the introduction of a child phospholipids in an amount that effectively reduces the incidence of necrotic enterocolitis.

 Typically, these phospholipids are administered to obtain a concentration between about 60 and about 2400 μmol, preferably between about 200 and about 1500 μmol, most preferably between about 400 and about 1000 μmol of phospholipids per kg per day. The source of phospholipids is not critical; phospholipids derived from egg lecithin are suitable for the present invention. Phospholipids are also readily obtainable from membranes of animal origin, including membranes of fat globules of milk. Other sources rich in phospholipids include soybeans and other vegetable oils. When using egg lecithin phospholipids, a preferred amount for enteral administration is an amount sufficient to produce at least 1.0 mg of long chain n-6 fatty acids per day. Preferably, the egg phospholipid provides arachidonic acid as a significant part of the n-6 fatty acids, and it is preferable that it also provides docosahexaenoic acid and / or other long-chain n-3 fatty acids in the above ratios. This may have a synergistic effect. However, it has been observed that the length and saturation of the fatty acids associated with the glycerol skeleton are not critical in this “phospholipid” aspect of the invention, and fatty acids other than long-chain PUFAs can be used in this case.

 In another aspect, the invention provides a method of reducing the incidence of necrotic enterocolitis in children. This method involves administering choline to the child in an amount effective to reduce the incidence of necrotic enterocolitis. Typically, said choline is administered to obtain a concentration between about 60 and about 1800 micromoles, more preferably between about 150 and about 1200 micromols of choline per kg per day. The source of choline is not critical; phosphatidylcholine may be preferred, and phosphatidylcholine derived from egg lecithin is suitable for the present invention. Other sources rich in choline or phosphatidylcholine include soybeans and other vegetable oils. When using choline from egg lecithin, it is preferable to administer it in combination with n-6 and / or n-3 fatty acids to produce at least 1.0 mg of long-chain n-6 fatty acids per day. Preferably, egg lecithin provides arachidonic acid as a significant part of n-6 fatty acids, and it is preferable that it also provides docosahexaenoic acid and / or other long-chain n-3 fatty acids in the above proportions. This may have a synergistic effect. However, from the point of view of the phospholipid aspect, the length and saturation of fatty acids associated with the glycerol skeleton of any phosphatidylcholine used in the invention are not critical, and in this case it is possible to use fatty acids other than long-chain PUFAs. Alternatively, choline from sources other than phospholipids may be used.

 In addition, since beneficial effects have been observed in children susceptible to enterocolitis, it is possible that these beneficial effects also extend to adults. Thus, a further aspect of the invention is the use of the above compositions for the treatment or prophylaxis of ulcerative colitis and related intestinal conditions in adults. In this case, the dosage will be determined based on the higher body weight of the adult patient and other factors known in the art.

Other aspects of the invention, including enteric preparations and methods for their preparation, are described further in the application. For example, in another aspect of the invention, a method for producing an enteral composition comprising egg yolk phospholipids comprises the following steps:
(a) obtaining a dry powder of egg phosphatides, practically free of cholesterol;
(b) dispersing said fraction of phospholipids in the aqueous phase to form a dispersion of phospholipids and
(C) mixing the specified dispersion of phospholipids with a thick suspension of other components of this enteral composition.

According to the method, it is preferable that the dispersion in the aqueous phase contains from about 2 to about 15 wt.% Egg phosphatide, and it is preferable to add egg phosphatide powder to water at a temperature of from about 20 to 50 ^° C. This aspect can be used to obtain the composition for baby food containing arachidonic acid and docosahexaenoic acid in the form of phospholipids, the specified enteral composition is obtained by this method.

 In yet another aspect, the invention provides a baby food preparation comprising protein, carbohydrates and lipids, an improvement of which is characterized in that the lipid mixture contains medium chain triglycerides and egg phospholipid, wherein the level of egg phospholipid is from about 1 wt.% To about 40 wt.% from a mixture of lipids and this egg phospholipid practically does not contain cholesterol. Typically, the level of egg phospholipid is from 5 to 30% by weight of the lipid mixture and the concentration of arachidonic acid in the preparation is from about 10 to about 31 mg per 100 kcal. More preferably, docosahexaenoic acid is also included in the preparation at a concentration of from about 3 to about 16 mg per 100 kcal and the presence of arachidonic acid and docosahexaenoic acid in a ratio of from about 4: 1 to about 2: 1.

Information confirming the possibility of carrying out the invention
Basic terminology
Fatty acids are hydrocarbon chains of various lengths containing carboxylic acid at one end, which makes them to some extent polar and hydrophilic in this position, while, on the other hand, they remain hydrophobic to varying degrees depending on the length of the hydrocarbon chain. Fatty acids are divided into classes based on the length of the hydrocarbon chain. For example, chains containing less than 6 carbon atoms are considered “short” chains; chains containing about 6-18 carbon atoms are “medium” and chains of 20 or more carbon atoms are considered “long”. Fatty acids may also have one or more double bonds, which are “unsaturation” points in the hydrocarbon chain. As used in this context, the term "long-chain PUFA" means a fatty acid of twenty or more carbon atoms having at least two carbon-carbon double bonds (polyunsaturated). The number and position of double bonds in fatty acids is determined by nomenclature agreement. For example, arachidonic acid (“AK” or “ARK”) has a chain of 20 carbon atoms and 4 double bonds starting at the sixth carbon atom from the methyl end. As a result, it is designated as “C ₂₀ : 4 n-6”. Similarly, docosahexaenoic acid ("DHA") has a 22 carbon chain with 6 double bonds starting at the third carbon atom from the methyl end, and is denoted as "C ₂₂ : 6 n-3". Less common long-chain PUFAs are also known, some of which are shown in Tables 2 and 5 (below the solid dividing line).

 "Glycerides" are complex lipids having a glycerol skeleton forming esters with fatty acids. A "triglyceride" (i.e., "triacylglycerol") contains three esterified fatty acids, one for each hydroxyl site on the glycerol skeleton. Di- and monoglycerides contain respectively two or one esterified fatty acid. Phosphoglyceride (ie, “phospholipid” or “phosphatide” - the terms are used interchangeably) differs from triglycerides in that it contains a maximum of two esterified fatty acids, while the third position of the glycerol skeleton is esterified with phosphoric acid to form “phosphatidic acid”. Under natural conditions, phosphatidic acid usually binds to alcohol, which causes the presence of a strongly polar onset of the molecule. Two such alcohols, commonly found in vivo, are choline and ethanolamine. "Lecithin" is a phosphatidic acid bound to the amino alcohol "choline" and is also known as "phosphatidylcholine". Lecithins vary in the content of the fatty acid component, and their source may be, for example, eggs and soy. Cephalin (phosphatidylethanolamine), phosphatidylserine, and phosphatidylinositol are other phosphoglycerides.

 Phospholipids are usually found in the membranes of all living systems. Traditional sources of phospholipids are egg yolk and soybean oil. Phospholipids can also be obtained from the brain, kidneys, heart and lung of mammals, or from membranes of fat globules of milk. In addition, sources of microbial nature (unicellular oils), such as algae and mushroom oils, in particular for components of fatty acids AK and DHA phospholipids, can be used.

 Chicken eggs are a relatively rich source of lipids. Approximately 33% of the chicken yolk is lipid, approximately 67% of which is triglyceride, 28% is phospholipid, and the remainder is mainly cholesterol (wt.%). These figures are approximate and may vary to some extent depending on the feed, breed and conditions of the chicken. Of the phospholipid fraction, approximately 75% is phosphatidylcholine and approximately 20% is phosphatidylethanolamine. The structure of choline is approximately 15 to 30% of each phospholipid molecule, depending on the particular fatty acid attached. Thus, the choline content in egg phospholipid can vary from about 10 wt.% To about 25 wt. % or in terms of total egg lipid - from about 3 wt.% to about 7 wt.%.

Songs
The compositions used in the invention contain n-6 and / or n-3 long-chain PUFAs. The source of a long-chain PUFA is not critical. Known sources of long-chain PUFAs include fish oil or marine organisms oils, egg yolk lipid and phospholipids, unicellular organisms oils (e.g., algae and mushroom oils). From the prior art it follows that some sources are better than others in terms of obtaining large quantities of specific long-chain PUFAs. Other edible semi-purified or purified sources of long-chain PUFAs will be apparent to those skilled in the art. New sources of long-chain PUFAs can be created using genetic manipulations with vegetable and oilseeds, and the use of these recombinant products is also included in this invention.

 Long-chain PUFAs can be incorporated into the composition in the form of esters of free fatty acids, mono-, di- and triglycerides, phosphoglycerides, including lecithins, and / or mixtures thereof. The administration of long-chain PUFA in the form of phospholipids, in particular phosphatidylcholine, may be preferred. Currently, the preferred source, at least when it is processed in such a way that its organoleptic properties and cholesterol level are acceptable, is probably egg yolk phospholipids, possibly due to the high content of phospholipids and / or phosphatidylcholine associated with PUFA, obtained from eggs.

 Long-chain n-6 and / or n-3 PUFAs can be used in the form of a solution for intravenous (i.e., parenteral) administration, such as, for example, choline and phosphatidylcholine. The solution for intravenous administration will preferably contain effective amounts of PUFA, phospholipid and / or choline in an acceptable volume of parenteral solution for daily administration. The exact concentration therefore varies widely depending on the intended volume of administration and is significantly higher in a small volume bolus or parenteral product than in a hydrated or nutritious parenteral product. Parenteral compositions will typically include pharmaceutically acceptable carriers and excipients such as buffers, preservatives, and the like.

 Long-chain n-6 and / or n-3 PUFAs, as well as choline and a phospholipid, can alternatively be administered in the form of an enteral composition. Enteral compositions containing long-chain PUFA, choline or phospholipid may be in the form of a solution or emulsion of the active ingredient or in a nutritional basis containing protein, carbohydrates, other fats, minerals and vitamins. Enteral compositions containing active ingredients can serve as either complementary or complete artificial nutrition. The concentration of long-chain PUFAs in the enteric composition can vary from almost 100 wt.% (In the case of a bolus emulsion) to 0.5 wt.% (In the case of a complete nutritional composition) of the composition depending on the route of administration and the intended purpose. In complete nutritional formulations, the concentration may even be lower with the introduction of a sufficient amount of formulation to deliver an effective amount of long-chain PUFA.

 A particularly preferred embodiment of the invention relates to an improved complete nutritional composition suitable for feeding babies, including premature babies. This preferred composition contains protein, carbohydrates and lipids, in which from about 6 to about 40 wt. % of the total lipid content is represented by egg phospholipid, which practically does not contain cholesterol. The term "practically does not contain" means that the cholesterol content in egg phospholipid does not exceed 0.1 wt.% And preferably less than 0.05 wt.% Of the total lipid content.

 Specialists will easily understand what “baby formula” means. In diluted or reconstituted (if it is initially in the form of a concentrate or powder) to a ready-to-feed state, a typical baby food composition contains approximately 10-35 g of protein per liter of composition, 20-50 g of lipid per liter of composition, 60-110 g of carbohydrates per liter of composition and other various components, such as vitamins, minerals, fiber, emulsifiers, etc. In order to explain the components of the composition for baby food and methods for its preparation, the following US patents are incorporated by reference: 1) US patent 5492899 issued by Masor et al .; 2) US patent 5021245 issued by Borschel et al .; 3) US patent 5234702 issued by Katz et al .; 4) US patent 5602109 issued by Masor et al. and 5) U.S. Patent 4,670,268 issued to Mahmoud. In particular, this embodiment of the invention encompasses an infant formula containing about 40-50 g of lipid per liter of composition, wherein the lipid contains a mixture of medium chain triglycerides and egg phospholipid, which is substantially free of cholesterol. Typically, the lipid mixture contains from about 1 to 40 wt.%, More preferably from about 5 to about 30 wt.% Egg phospholipid. This embodiment is specifically designed to produce long-chain PUFAs selected from n-3 fatty acids and n-6 fatty acids, phospholipids and / or choline in amounts suitable for children.

Production method
Since chicken egg yolks include both triglycerides and phosphatides, it may be preferable to treat the egg yolks with organic solvents so as to separate phosphatides from triglycerides, sterols (e.g. cholesterol) and other components. Various methods described in the literature are suitable for this department, at least at the laboratory level. Alternatively, these cholesterol-free egg phosphatides are commercially available in dry powder form from Pfanstiehl, Inc. (Waukegan, IL), Catalog No. P-123.

Egg phosphatide is then introduced into the enteric composition of the invention. Due to the lipid content, it was expected that the introduction of egg phosphatides into the enteral composition is easier to carry out in the oil phase. However, it was unexpectedly discovered that the lipid-lipid dispersion data were unacceptable and that the preparation of an aqueous dispersion of egg phosphatide resulted in an improved product. For best results, aqueous dispersions of about 2-15 wt.%, Preferably from about 3 to about 8 wt.% Should be prepared in cool to room temperature water (about 20-25 ^o C). Warmer water gave less acceptable organoleptic properties.

Thick suspensions of carbohydrate, protein, and lipid, which contain a macro food source, are prepared separately, as is known in the art, and these suspensions are mixed at about 130 to 140 ^° C. Immediately before homogenization, the phosphatide dispersion is mixed with the remaining components of the composition.

 In a particularly preferred embodiment, before adding the dispersion of phosphatide to the mixture of the final product (immediately before homogenization), the dispersion of phosphatide is deaerated under moderate vacuum. Deaeration can be carried out using any mechanism, but a spray deaerator at approximately 15 inches (375 mm) Hg provided satisfactory results. As shown, this additional step improves the organoleptic properties and the smell of the final product even more than filtering through activated carbon or a combination of these two methods (see Example 3).

 To obtain compositions for parenteral administration used in this invention, can be used in the traditional technology for sterile parenteral preparations. In this case, it may be preferable not to use egg phosphatides and use triglyceride oils or fatty acid esters instead, such as can be found in recombinant or unicellular oil sources.

Industrial application
The compositions of this invention are used as artificial nutrition for children and / or adults. The addition of long-chain PUFAs, in particular, n-6 and n-3 fatty acids and especially AK and DHA, is generally considered favorable for the development of the nervous system and visual activity in children, although conflicting data have also been found in the literature.

Compositions used in this invention include compositions containing any or all of the following components:
(a) a long-chain PUFA selected from n-6 and n-3 fatty acids, usually in the form of a phospholipid or phosphatidylcholine;
(b) polar phospholipids, regardless of the nature or length of the attached fatty acids;
(c) choline, preferably phosphatidylcholine.

 For example, a composition enhanced with egg phospholipid, described in detail in the examples, provided elevated levels of the described components and was unexpectedly found to significantly reduce the incidence of NEC in populations of children susceptible to NEC. In a more specific embodiment, a method for reducing the incidence of NEC is carried out by administering arachidonic acid (AK, 20: 4 n-6) or, more preferably, AK in combination with docosahexaenoic acid (DHA, 22: 6 n-3).

In a broader sense, this aspect of the invention encompasses a method of reducing the incidence of necrotic enterocolitis in children who are susceptible to necrotic enterocolitis. The method comprises administering an effective amount of at least one PUFA selected from the group of C ₂₀ n-6 fatty acids, C ₂₂ n-6 fatty acids, C ₂₀ n-3 fatty acids and C ₂₂ n-3 fatty acids. The introduction is carried out at a level of at least 1.0 mg of n-3 fatty acids per kilogram of body weight per day. In a more preferred embodiment, a combination of n-6 and n-3 fatty acids is used in a weight ratio of from about 2: 1 to about 4: 1.

 The following discloses a method of reducing the incidence of necrotic enterocolitis in children. This method involves administering to a child egg phospholipids in an amount providing at least 1.0 mg of long chain n-6 fatty acids per day. Preferably, egg phospholipids provide AA in a significant proportion of n-6 fatty acids and preferably also provide DHA and / or other long-chain n-3 fatty acids in the above proportions.

 An additional aspect of this invention relates to the enteral administration to humans of phospholipids, in particular phospholipids containing AK and / or DHA, which rapidly increase the levels of fatty acids AK and DHA in human serum relative to compositions containing triglycerides AK and DHA.

 A more suitable measure for administering the compositions of the invention is the daily intake in mg / kg of the child’s body weight. Table 1 provides recommendations for the minimum, preferred, and ideal ranges for each of the compositions used in the invention.

 The wide range of ranges exists largely due to the fact that not all children who are likely to benefit from this invention will consume equal amounts of the composition. Those who consume less receive less than each ingredient. The ideal consumption range suggests that approximately 100 kcal will be consumed. Controversial methods for assessing choline are also controversial.

 As can be seen from Table 1, an approximately 2-4-fold excess of AA relative to DHA is most preferred. It is also shown that the minimum levels of phospholipid and choline are identical. This is achieved by obtaining the entire phospholipid in the form of phosphatidylcholine. Since other nitrogen-containing alcohols replace choline (for example, ethanolamine, serine or inositol), the relative amount of choline in the total phospholipid is reduced.

AK and / or DHA can be administered individually as separate components, either together or in combination with other ingredients, such as protein, lipid, carbohydrate, vitamins and minerals. Artificial nutrition for babies with low birth weight is either parenteral (intravenous feeding) or enteral. Thus, appropriate levels of long-chain PUFAs can be introduced into a parenteral nutrient solution or added to a given enteral formulation for low birth weight babies. Most preferably, the method of the invention is carried out by enteral administration of an infant formula formulated for low birth weight babies that contains AK and DHA. This baby food composition further contains suitable levels of hydrocarbon and protein and a suitable combination of minerals and vitamins. An example of a composition for infant food for use in the methods of the invention is a modified ^{Similac Special Care ® (Ross Products Division} of Abbott Laboratories, Columbus, Ohio), which is discussed in more detail in Example 2.

 An additional aspect of the present invention is a method of increasing levels of arachidonic acid and docosahexaenoic acid in human serum. This method involves the step of administering to a specified person an enteral composition containing AK and DHA in the form of phospholipids.

 Recent studies of these applicants have shown that the administration of long-chain PUFAs to children susceptible to NECs will reduce the incidence of NECs and may also reduce the level and severity of NECs. Applicants have also found that administration of animal or plant phospholipids also effectively reduces the incidence of NEC in populations of children that are susceptible to NEC.

Example 1
Egg yolk phospholipid is obtained from Pfanstiehl, Inc. (Waukegan, IL-Catalog No. P-123) and used in the following Examples. The fatty acid and cholesterol profiles of this egg phosphatide are presented in Table 2. The sum of all n-3 and all n-6 long-chain PUFAs is also shown.

 Specialists will appreciate that the specific levels of various fatty acids contained in egg yolk lipid vary depending on the breed, feed and age of the chickens. In addition, the extraction method used by Pfanstiehl to produce the phosphatide used in the Examples provides a material that contains extremely low cholesterol, while providing a fatty acid profile that is widely used in food preparation.

Example 2
In this example, the "Experimental" and "Control" baby food formulations are prepared respectively with and without the addition of egg phosphatide from Example 1. The control composition is Similac Special Care ^® (Ross Products Division of Abbot Laboratories, Columbus, Ohio), it is prepared using the following list of ingredients, which gives the composition having the composition shown in Tables 3-5: water (Kosher), non-fat milk, hydrolyzed corn starch, lactose, fractionated coconut oil (medium chain triglycerides), protein concentrate mol whey, soybean oil, coconut oil, tribasic calcium phosphate, potassium citrate, sodium citrate, magnesium chloride, ascrobic acid, mono- and diglycerides, soya lecithin, calcium carbonate, carrageenan, choline chloride, ferrous sulfate, m-inositol, taurine , nicotinamide, L-carnitine, α-tocopheryl acetate, zinc sulfate, calcium pantothenate, potassium chloride, cupric sulfate, riboflavin and vitamin A palmitate hydrochloride, thiamine chloride hydrochloride, pyridoxine, biotin, folic acid, manganese sulfate, phylloquinone, vitamin D ₃ , selenite on tria and cyanocobalamin.

 Typically, thick suspensions of protein, carbohydrate, lipid, vitamins and minerals are prepared separately and then mixed before homogenization, as is mainly described in previously included US Patents relating to the manufacture of baby food formulations.

In the experimental composition, egg phosphatide corresponding to Example 1 is introduced into the composition during preparation. First, egg phosphatide is dispersed in water at 25 ^{° C.} to obtain an 8% dispersion. Immediately prior to homogenization, the phosphatide dispersion is mixed with thick suspensions of protein, carbohydrate, vitamins, minerals and other lipids and an “Experimental” formulation is obtained having the composition shown in Tables 3-5. The amounts of each component are given both "per liter" and "per kcal", since it is well known in the prior art to prepare formulations for baby food with calories higher or lower than the standard of 20 kcal per fluid ounce (approximately 0.7 kcal / ml) .

 Table 4 shows the lipid content (the only difference) used in the Control and Experimental products. It can be noted that the two compositions have the same total amount of lipids, but differ fundamentally by replacing part of the medium chain triglycerides with egg phospholipid. This replacement provides significantly higher levels of phospholipids and choline, as well as additional long-chain PUFAs.

Table 5 shows the fatty acid profile for the Control and Experimental formulations. It represents the sum of the egg lecithin fatty acid components and the Similac Special Care ^® formulation.
The inclusion of egg phosphatide results in a 0.21 wt.% N-3 long chain fatty acid content in the total lipid mixture and 0.48 wt.% N-6 long chain fatty acid in the total lipid mixture. In particular, 0.14 wt.% Of the total lipid mixture is DHA and 0.41 wt.% Of the total lipid mixture is AA. When administered 100 kcal / kg / day per 1 kg of child’s body weight, this formula provides approximately 22 mg of AA and approximately 7 mg of DHA per day. Of course, this is only one of the possible embodiments of the ratios in accordance with this invention.

Example 3
In this experiment, variants of the method are evaluated in terms of reducing organoleptic disadvantages associated with the use of egg phospholipids. Isolation of egg phospholipids used in this invention often leads to egg phosphatide, which has any organoleptic properties unsuitable for use in infant formula. They can be improved upon further receipt of a product that is not unpleasant for either the child or the caregiver. The following describes a method for improving the final product.

A number of nutritional formulations are prepared similar to the composition corresponding to Example 2, except that 6 wt.% Of the fat mixture is egg phospholipid, which is pretreated using various procedures. Egg phospholipid is dispersed in part of the oil mixture described in Example 2, or in part of water. Oil dispersions are unacceptable and cannot be used even after heating to about 95 ^° C. A dispersion of a phospholipid in water at room temperature to warm is easy to prepare, and it is the preferred means for preparing an aqueous dispersion.

An initial portion of a dispersion of 3 wt.% Egg phospholipid is prepared by mixing the phospholipid with water at 90 ^{° C.} for about 1 hour. Part of this dispersion is carried out through: (1) only a deaerator; (2) only a device for filtering through activated carbon; (3) the combination of a deaerator and a device for filtering through activated carbon; and (4) are not processed.

 A device for filtering through activated carbon contains 80 g of activated carbon, and the device for deaeration operates under moderate vacuum (15 inches (375 mm) Hg). Portions of the portion are passed through the filtering device 3 times and once through a deaerator. When using both methods, part of the mixture is first passed through a filter, and then through a deaerator. The processed portions of the mixture are then added to the appropriate nutritional formulations immediately before the homogenization and packaging of the samples.

 Then the samples are first evaluated for taste (organoleptic properties) with the help of experts. The evaluation results are presented in Table 6.

 Unexpectedly, the sample with the smallest taste is the sample containing the dispersion, which is carried out only through the deaerator. The dispersion, which is carried out through a deaerator and an activated carbon filter, has the worst result with the exception of control (phospholipid dispersion without treatment).

Example 4
The formulations prepared in accordance with Examples 2 and 3 feed the children in a study conducted in the Newborn Care Unit of the Center for Newborns of the University of Tennessee, led by Dr. Susan E. Carlson with financial support from the Ross Products Division of Abbot Laboratories (Study AE78), grant NICHD RO1-HD31329 and a grant from the National Eye Institute RO1-EY08770. Research options include growth, development of the nervous system, and visual activity. It is believed that PUFAs are physiologically important for the development of the brain and eyes and quickly accumulate in the fetal tissues in the last trimester of pregnancy. Thus, premature babies do not accumulate normal levels of long-chain PUFAs compared to full-term babies.

 Inclusion criteria: Inclusion in this clinical study is based on "low" birth weight (less than 1500 g (in the range of 750-1375 g)) in the absence of a diagnosis of heart, respiratory, gastrointestinal or other systemic diseases. The child also does not have asphyxia or clinical complications in the history of birth regarding blood group incompatibility. Mothers included in the study of children have no prenatal infections in the medical record with proven adverse effects on the children. Maternal addiction is an exclusion criterion. All children begin to be fed orally on the 7th day of life.

 During the clinical study, it includes only 120 children in the first 7 days of life. With the exception of one child who is transferred to another hospital shortly after inclusion in the study (Control), all other children (n = 119) are monitored in the same hospital. Children are included (in a random "blind" way) in 1 of 3 groups, two of which receive the Control team during their stay in the hospital, and one group receives the Experimental staff (see Example 2). Children lost (deceased) during their stay in the hospital are replaced by other children included in the same treatment group. According to the research plan, a larger number of children are fed with the Control. The total number of children fed with the Control Panel is more than two times the number of children fed with the Experimental Panel.

 Results: Surprisingly, an increased incidence of necrotizing enterocolitis (NEC) was observed in the Control Groups compared with the Experimental Group. Table 7 groups the total number of newborns in accordance with the treatment (Control in relation to the Experiment) and presents the number of newborns in each group who develop NEC. NEC is believed to be present or suspected when clinical signs and symptoms are consistent with the disease, for example, bloating, residual masses in the stomach, bile vomiting, feces with blood, mucus in the stool and the presence of C-reactive protein at a level of ≥ 0.5 mg / decal (Pourcyrous et al. "The value of serial reactions of C-reactive protein in neonatal infections and other diseases" Pediatr., 1993, T. 92, S. 431-435). NEC is confirmed in 15 children in the Control and only 1 in the Experimental group.

 Statistical analysis of these results using the Fisher test (double-tailed) shows that the number of children with confirmed NEC in the Control group (s) significantly exceeds (p = 0.039) the number of children with NEC in the Experimental group.

Example 5
In this experiment, the incorporation of AK and DHA with parenteral nutrition (intravenous nutrition) is evaluated. A solution for parenteral administration may contain various components known in the art, with the addition of AA and DHA in the form of a phospholipid, triglycerides or methyl esters. AK and DHA may be the only active ingredients mixed with accepted parenteral carriers or excipients, or, more preferably, AK and DHA are included in the composition for parenteral administration, intended to supplement or provide complete artificial nutrition of the child. Typical parenteral nutritional solutions contain lipid levels that provide approximately 2g / kg / day. The level of AK and DHA in the lipid mixture should preferably allow administration of 10 to 30 mg / kg / day AK and 3 to 15 mg / kg / day DHA.

Example 6
In this experiment, egg lecithin of an experimental composition corresponding to Example 2 is replaced with soya lecithin at a level approximately 10 times its level in the control composition. Soya lecithin, like other phospholipids isolated from plant sources, does not contain long-chain polyunsaturated acids, but the polar nature of phospholipids and their ability to quickly incorporate into the intestinal mucosa can create a protective effect on the lining of the intestine, which leads to results comparable to the results obtained for the experimental composition corresponding to Example 2. In addition, soya lecithin contains linoleic acid (18: 2n-6 - the precursor of food basic fatty acid A ) And linolenic acid (18: 3n-3 - a food precursor essential fatty acids DHA).

Example 7
In this experiment, the use of phospholipids containing AK and DHA in the composition for baby food is compared with triglycerides containing AK and DHA. The composition corresponding to Example 2 is compared with a similar composition for baby food, in which egg phospholipid is replaced by a mixture of triglycerides of unicellular microorganisms containing comparable levels of AK and DHA.

 The clinical evaluation includes healthy full-term infants in order to determine serum levels of AK and DHA after enteral administration. It is expected that in children fed a composition with phospholipids, serum levels of AK and DHA will be achieved closer to these levels in children fed breast milk, compared with a control composition containing AK and DHA in the form of triglycerides. This experiment should demonstrate that phospholipids containing AA and DHA are the preferred form of administration compared to triglycerides containing AK and DHA. Thus, in this context, improved enteral formulations and methods for increasing serum AK and DHA levels are provided.

 Modifications and alternative embodiments of the compositions and methods of this invention will be apparent to those skilled in the art in light of the foregoing description. Accordingly, this description is intended for illustration only and is intended to provide professionals with a way to implement it. The full scope of the invention, the protection of which is provided, is defined in the attached claims.

Claims (28)

 1. A method of reducing the incidence of necrotic enterocolitis in children, characterized in that the child susceptible to necrotic enterocolitis is administered at least one n-6 polyunsaturated fatty acid in an amount of from about 1.0 mg / kg of the body weight of the child to about 60 mg / kg per day, in combination with at least one n-3 polyunsaturated fatty acid, taken in an amount of from about 0.25 to about 35 mg / kg per day.  2. The method according to p. 1, characterized in that arachidonic acid is used as at least one n-6 polyunsaturated fatty acid.  3. The method according to p. 2, characterized in that the introduction is carried out enterally.  4. The method according to p. 2, characterized in that the introduction is carried out parenterally.  5. The method according to claim 1, characterized in that docosahexaenoic acid is used as the indicated n-3 polyunsaturated fatty acid.  6. The method according to claim 1, characterized in that arachidonic acid is used as the indicated n-6 polyunsaturated fatty acid, and docosahexaenoic acid is used as the indicated n-3 polyunsaturated fatty acid.  7. The method according to claim 6, characterized in that the introduction is carried out by feeding a sufficient amount of an enteral composition containing arachidonic acid and docosahexaenoic acid, and at the same time from about 1.0 to about 60 mg / kg per day of arachidonic acid is administered to the specified child and from about 0.25 to about 35 mg / kg per day of docosahexaenoic acid.  8. The method according to claim 7, characterized in that the mass ratio of arachidonic acid and docosahexaenoic acid is selected in the range from about 2: 1 to about 4: 1.  9. The method according to claim 1, characterized in that said n-6 polyunsaturated fatty acid and said n-3 polyunsaturated fatty acid are long chain fatty acids, and they are independently selected from at least one source selected from the group consisting of egg lecithin, mushroom oils, algae oils and marine organisms oils.  10. The method according to claim 1, characterized in that said n-6 polyunsaturated fatty acid and said n-3 polyunsaturated fatty acid are used in the form of phospholipids.  11. The method according to claim 5, characterized in that they use a solution for parenteral administration containing at least 20 mg / l of arachidonic acid and at least 10 mg / l of docosahexaenoic acid.  12. The method according to p. 11, characterized in that the solution contains approximately 20-200 mg / l of arachidonic acid and approximately 10 mg to 50 mg / l of docosahexaenoic acid.  13. The method according to claim 1, characterized in that the specified child is fed a sufficient amount of an enteral nutritional composition containing protein, carbohydrate and phospholipids, and at least 2.0 mg / kg n-6 polyunsaturated fatty acids are introduced into day and at least 0.5 mg / kg n-3 polyunsaturated fatty acids per day.  14. The method according to item 13, wherein at least 2.0 mg / kg of arachidonic acid and at least 0.5 mg / kg of docosahexaenoic acid are administered per day.  15. The method according to 14, characterized in that used for feeding an enteral composition containing egg phospholipid.  16. A method of reducing the incidence of necrotic enterocolitis in children, characterized in that a child susceptible to necrotic enterocolitis is administered an effective amount of phospholipids daily, ranging from about 60 μmol / kg of the body weight of the child to about 2400 μmol / kg per day.  17. The method according to clause 16, wherein the introduction is carried out by means of a sufficient amount of a nutritional composition.  18. The method according to 17, characterized in that by means of a sufficient amount of the specified nutritional composition is administered between approximately 200 and approximately 1500 μmol / kg phospholipids per day.  19. The method according to clause 16, wherein said phospholipids are obtained from egg lecithin.  20. The method according to 17, characterized in that the phospholipids are administered in combination with at least one polyunsaturated fatty acid selected from the group consisting of arachidonic acid and docosahexaenoic acid.  21. The method according to claim 20, characterized in that from a combination of these phospholipids with arachidonic acid and docosahexaenoic acid, from about 200 to about 1500 μmol / kg phospholipid, from about 5.0 to about 40 mg / kg arachidonic acid, and from about 1.5 to about 20 mg / kg of docosahexaenoic acid per day.  22. A method of reducing the incidence of necrotic enterocolitis in children, characterized in that a child susceptible to necrotic enterocolitis is administered an effective amount of choline daily from about 60 to about 1800 μmol / kg choline per day.  23. The method according to p. 22, characterized in that the introduction is carried out by means of a nutritional composition containing the specified choline in sufficient quantity.  24. The method according to p. 23, characterized in that by means of a sufficient amount of the specified nutritional composition is administered between approximately 150 and approximately 1200 μmol / kg choline per day.  25. The method according to item 22, wherein the specified choline is used in the form of phosphatidylcholine.  26. The method according A.25, characterized in that said phosphatidylcholine is isolated from egg lecithin.  27. The method according to item 23, wherein said choline is administered in combination with at least one polyunsaturated fatty acid selected from the group consisting of arachidonic acid and docosahexaenoic acid.  28. The method according to item 27, wherein by combining the specified choline with arachidonic acid and docosahexaenoic acid from about 150 to about 1200 μmol / kg of choline, from about 5.0 to about 40 mg / kg of arachidonic acid and from about 1.5 to about 20 mg / kg of docosahexaenoic acid per day. Priority on points:
10/03/1997 - according to claims 1, 5, 7, 9-14 and 16-28;
02.21.1997 - according to claims 2-4;
03/28/1997 - according to paragraphs 6, 8 and 15.

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